Wood is a complex material which is composed of cellulose, hemicellulose and lignin along with other minor components. Lignin is associated with cellulose and hemicellulose, and is probably covalently bound to both cellulose and hemicellulose.
In the paper-making process, lignin is generally removed from the wood pulp since it lends a brownish color, reduces strength and imparts other undesirable characteristics to the finished product. Removal of lignin can be achieved in many ways.
A majority of the lignin is initially removed from wood pulp through chemical pulping (e.g. kraft process). In the subsequent bleaching process, chemical pulp is routinely reacted with chlorine and other delignifying chemicals to further remove lignin and then reacted with bleaching agents to modify the lignin from pulp, providing a stable brightened pulp. However, the treatment with chlorine is undesirable from an environmental standpoint because the resulting effluents contain a large number of toxic compounds (e.g. chlorinated phenolics). Concern about the environmental harmful effects caused by pulp bleaching with chlorine containing chemicals has driven the industry to seek alternative bleaching methods.
Attempts to use enzymes derived from fungal and bacterial sources to enhance delignification and brightening, while lowering or eliminating the use of chlorine chemicals have been described in the literature. However, very few enzyme systems have been found which selectively act on pulp but do not adversely affect the cellulosic content of pulp.
Xylanases are hemicellulase enzymes that catalyze the hydrolysis of xylan, a major component of hardwood and softwood hemicellulose, and are usually associated with the cellulose and lignin components of plant cell walls. Xylanase has proven to be a valuable enzyme for the pre-bleaching of pulp to enhance delignification of wood pulp by facilitating the removal of lignin from pulp. A proposed mechanism for this action is that during kraft pulping, xylan is first solubilized in the cooking liquor. In the later stages of the cook xylan is reprecipitated on the pulp fibres. When xylanases are used in the pre-bleaching of pulp, partial hydrolysis of these reprecipitated xylan fractions renders the pulp surface more permeable for lignin removal. Therefore, xylanase pre-bleaching results in the use of lower amounts of bleaching chemicals as compared to nonenzymatic bleaching. Most of the enzyme preparations initially described in the literature are active at acidic pH ranges with optimal temperatures reaching 50.degree. C.
For industrial application, especially in the pulp bleaching industry where the processes take place at high temperatures and alkaline pH, it would be significantly advantageous if xylanases were available which are active at high temperatures over a wider pH-range, especially pH 7-10, than are now currently available.
The xylanases purified from Microtetraspora flexuosa are excellent candidates in the pre-bleaching of pulp because they are active at high temperatures and alkaline pH, and they act on the hemicellulose/cellulose matrix of the pulp with which the lignin is associated or bound, such that after enzyme treatment, the lignin is released and/or rendered releasable by an appropriate extractant.
Recently, several thermophilic xylanases from fungal and bacterial microorganisms have been identified. For example, a thermophilic xylanase has been isolated from Actinomadura reclassified as Microtetraspora having an optimal pH of 6.0-7.0 and temperature range of 70.degree. to 80.degree. C. (Holtz, C. et al Antonie van Leewenhoek 59:1-7, 1991). EP 0473545 discloses that the bacterial strain Thermomonospora fusca produces thermostable xylanases active at temperatures 10.degree.-90.degree. C., preferably, 50.degree.-80.degree. C. over a wide pH range, i.e., from about 5-10, with the more preferred range between 6.6-9.5. In addition, WO92/18612 discloses a xylanase enzyme derived from the genus, Dictyoglomus, having activity over a broad pH range (5.0-9.0) and thermostability at temperatures ranging from 60.degree. to 90.degree. C.
Although thermostable xylanases active in the alkaline range have been described in the literature, the need still exists to identify novel xylanases that are more efficient in applications relating to delignifying and brightening of pulp compared to conventional bleaching agents and xylanases now available. Moreover, at the time of Applicants' invention, multiple xylanases from Microtetraspora flexuosa were unknown to exist that have optimal xylanase activity in the alkaline range.